In the prior art, scanning microscopes have been proposed which are used, for example, in fluorescence microscopy to excite dyes by laser light to emit fluorescent radiation, which is then captured by a detector to form an image of the object to be examined. The microscopes used in this area of microscopy are, in particular, scanning confocal microscopes which, unlike standard microscopes, do not illuminate the entire object at a particular point in time, but produce a typically diffraction-limited spot of light with which the object is scanned point by point. The light signals detected by the detector at the individual object points are then combined to form a complete image of the object.
Such a confocal microscope typically includes a scanning device of the type called a point scanner, which directs the illuminating light beam emitted by the light source into the entrance pupil of the illumination optics. The illumination optics transform the illuminating light beam entering its entrance pupil into a focused light distribution, which will hereinafter be referred to as “illumination focus”. The shape and size of the illumination focus depend on the optical properties, particularly the numerical aperture of the illumination optics. If the illuminating light beam enters the entrance pupil of the illumination optics centrally and perpendicularly; i.e., along the optical axis, then the illumination optics produce an elongated illumination focus which has a smaller extent transverse to the optical axis than along the optical axis. Then, in order to scan the object, the illumination focus is moved transversely to the optical axis by the point scanner varying the angle of incidence at which the illuminating light beam enters the entrance pupil of the illumination optics. This can be accomplished using, for example, a movable mirror system.
Thus, in order to form a three-dimensional image using a confocal microscope, the object must be scanned point by point in the manner described above. Since this is a relatively complex process, a microscopy technique referred to in the literature as selective plane illumination microscopy (SPIM) was proposed recently. This technique uses an illumination objective and an observation objective, which are arranged at an angle of 90 degrees relative to each other. The illumination objective, in cooperation with a cylindrical lens located upstream thereof, produces an approximately two-dimensional distribution of illumination light, which passes through the object along the optical axis of the illumination objective. Such a light distribution is frequently also referred to as “light sheet” or “light disk”. The target region of the object that is illuminated by this light sheet is imaged by the observation objective onto a detection surface, such as a CCD, the optical axis of the observation objective being perpendicular to the light sheet. If the object is then moved through the light sheet, it is possible to acquire tomographic images of the object using this configuration. In order to produce as thin a light sheet as possible, the illumination objective must have a correspondingly high numerical aperture. Moreover, the free working distance of the illumination objective must be large enough to prevent collision with the observation objective. Accordingly, the numerical aperture of the illumination objective determines the thickness of the light sheet, and thus, the optical resolution along the optical axis of the observation objective.
In a modified SPIM method described in WO 2010/012980 A1, illumination and detection are performed by one and the same objective. To this end, the entrance pupil of the objective is under-illuminated at an off-center position; i.e., the illuminating light beam passes through a portion of the entrance pupil that is transversely offset from the optical axis. A cylindrical lens upstream of the objective produces an illuminating light sheet in the object, which light sheet is inclined to the optical axis of the objective. The target region illuminated by this light sheet is then imaged by the objective.
All of the above-described systems use a cylindrical lens to obtain the desired oblique illumination of the object. However, the use of such a cylindrical lens has disadvantages. For example, these devices are designed exclusively for oblique illumination by means of a light sheet and do not allow for a different use, such as point-by-point confocal scanning. Moreover, it would be desirable to be able to vary the spatial distribution of the light intensity of the light sheet produced for oblique illumination. This is not possible using a cylindrical lens.
For further reading, reference is made to A. H. Voie et al.: “Orthogonal-plane fluorescence optical sectioning: three-dimensional imaging of macroscopic biological specimens”, Journal of Microscopy 170, 229-236 (1993); J. Huisken, J. Swoger, F. Del Bene, J. Wittbold, E. H. K. Stelzer: “Optical sectioning deep inside live embryos by selective plane illumination microscopy”, Science 305, 1007 (2004); F. Fahrbach, A. Rohrbach: “Microscopy with non diffracting beams”, FOM 2009, Krakau; C. Dunsby: “Optically sectioned imaging by oblique plane minor microscopy”, Optics Express Vol. 16, 25 (2008); DE 10 2005 027 077 A1; DE 44 16 558 C2; U.S. Pat. No. 5,952,668; WO 2006/127692 A2; DE 10 2006 021 317 B3; WO 2007/128434 A1; US 2009/01354342 A1; DE 10 2008 024 568 A1; US 2008/0032414 A1.